PROJECT SUMMARY Accurate identification of the replication-competent HIV-1 latent reservoir provides a basis for the design of any cure strategy; however, this is challenging because of its sparse distribution. Further, HIV-1 DNA exists in different forms and proviruses are genetically variable. The recently developed Full-Length Individual Proviral Sequencing (FLIPS) assay allows for the identification of genetically intact and potentially replication-competent HIV-1. Using FLIPS, 5% of the proviruses were identified as genetically intact and potentially replication competent in participants on effective long-term (3-17 years) antiretroviral therapy (ART). In addition, intact proviruses were enriched in effector memory CD4+ T cells, and multiple intact proviruses were found to be identical, suggesting a role for cellular proliferation in the maintenance of the latent HIV-1 reservoir. We have reported that HIV-1 episomal 2-long terminal repeat circular DNA (2-LTR circles), which are likely dead ends in the viral replication cycle in vivo, persist at relatively high levels for at least 3 years after initiation of ART containing an integrase strand transfer inhibitor (INSTI) during primary or chronic HIV-1 infection. Since INSTIs are now recommended in all guidelines as essential components of initial regimens, most infected individuals initiating contemporary ART may carry persistent levels of 2-LTR circles. It is therefore important to clarify whether genetically intact HIV-1 2-LTR circles are present in individuals who commenced INSTI-containing ART, because their persistence can interfere with the accurate quantification of the genetically intact and most likely replication-competent reservoir. We will therefore answer three important questions in the proposed study: (1) Do persisting HIV-1 2-LTR circles contain genetically intact HIV-1 genomes in CD4+ T cells during the first 3 years of INSTI-containing ART? If yes, at what level compare to genetically intact linear/proviral HIV-1? (2) How do genetically intact, replication-competent proviral forms distribute in CD4+ T cell subsets prior to and following initiation of INSTI-containing ART? Does this distribution change during the first 3 years of therapy? (3) Is HIV-1 replication occurring during the first 3 years of INSTI-containing ART and how do 2-LTR and integrated viral sequences relate to each other and evolve over time? This will be the first characterization of HIV-1 DNA, that accurately accounts for the linear/proviral forms and 2- LTR circles following initiation of treatment with INSTI-containing ART, which is now standard of care in many jurisdictions. Persistence of genetically intact 2-LTR circles will cause an overestimation of the potentially replication-competent latent HIV reservoir even when near full-length sequencing techniques are employed. This will also be the first longitudinal assessment of the landscape of genetically intact provirus and 2-LTR circles and how these evolve within CD4+ T cell subsets during early (0-3 years) therapy. This will inform the potential targeting of cure strategies. Finally, it will clarify whether ongoing viral replication maintains the latent reservoir during the first months of therapy, or whether this is driven by cellular proliferation even at these early time points.